Our long term goal is to elucidate the structure and genesis of defective interfering (DI) influenza virus, the mechanism of DI influenza virus mediated interference and hopefully, the role of the DI virus in the evolution and the diversity of influenza viruses in nature. In this present project our specific objectives are to further investigate the structure of DI RNA and its relationship to progenitor RNAs, to investigate the transcription and translation products of DI RNA in vitro and in infected cells, to establish an in vitro system of influenza viral RNA replication, and finally to determine the role of DI viruses viral persistency and diversity. DI virus from influenza A, B and C type viruses will be produced by passing virus at a high multiplicity using productive cell-virus systems. DI viruses will be amplifed using homologous standard virus as helper. Detailed structure analyses of DI RNA and their relationship to the progenitor genes will be performed by DNA cloning, heteroduplex mapping, restriction analyses and sequence determination. The mechanism of DI virus mediated interference will be elucidated by studying the transcription and replication of DI viral RNA in vitro as well as in infected cell. Subsequently, the effect of DI virus on the transcription and replication of standard viral RNAs will be determined both in vitro replication and transcription systems as well as in cells coinfected with both DI virus and standard virus. The role of DI viruses will be investigated by isolating variant viruses resistant DI mediated interference and determining the gene(s) responsible for the resistance.